Method for measuring the association of substructures of pathological protein depositions

ABSTRACT

A method for the diagnostic detection of diseases associated with protein depositions (pathological protein depositions) by measuring an association of substructures of the pathological protein depositions, structures forming pathological protein depositions, structures corresponding to pathological protein depositions and/or pathological protein depositions as a probe; to substructures of the pathological protein depositions, structures forming pathological protein depositions, structures corresponding to pathological protein depositions and/or pathological protein depositions as targets; characterized in that the target is detected in liquid phase wherein, in the case of detecting Alzheimer&#39;s disease, the liquid phase is obtained from body fluids or is itself a body fluid; with the proviso that the association of the probe to the target is measured before self-aggregation of the probe predominates.

[0001] The present invention relates to a method for the diagnosticdetection of diseases associated with pathological protein depositions.

[0002] A number of diseases is associated with the occurrence ofpathological protein depositions. It is often unclear whether theprotein depositions are only manifestations of a clinical picture, orwhether such protein depositions themselves are the pathogens and thusthe cause of the disease. Thus, neurodegenerative diseases are known inwhich, for example, protein depositions referred to as amyloid plaquescan be detected in the brain of afflicted persons. Such diseasesinclude, for example, Alzheimer's disease, bovine spongiformencephalopathy (BSE), Creutzfeldt-Jakob disease (CJD), laughing deathsyndrome, scrapie, and possibly other diseases which were referred to as“slow virus” diseases in the past. More recently, the BSE disease, inparticular, has become a focus of public attention, which is due to thefact, inter alia, that BSE has been connected with the Creutzfeldt-Jakobdisease in humans. Today, the mechanisms by which the proteindepositions affect the pathological process are still unclear. Therelationship, observed by Prusiner, between infectiosity and theconcentration of certain proteins which play a role in the pathologicalprocess of scrapie, a neurode-generative disease in sheep, isremarkable. Pathological protein depositions appear not only in diseasesof the neuronal system, but are observed in other organs as well, suchas in a disease of diabetes type II.

[0003] A survey of prion diseases has been published by D. Riesner in“Chemie in unserer Zeit” (1996), p. 66-74. Inter alia, it is set forththerein that a reliable and quick diagnosis is a priority problem ofprion research, not only to ensure biological safety, but also topromote the basic research which involves a lot of open questions as tothe replication and pathogenesis of prions. Especially for Alzheimer'sdisease, the pathological picture has been described relatively well.“Senile plaques”, which substantially consist of aggregated amyloid-βprotein, and “paired helical filaments”, which are constituted ofabnormally altered tau protein, are closely connected with Alzheimer'sdisease. The present state of the art of the biochemical diagnosis ofAlzheimer's disease is the immunological concentration measurement ofsoluble Aβ peptides (Motter et al., reduction of β-amyloid peptide₁₋₄₂in the cerebrospinal fluid of patients with Alzheimer's disease, Ann.Neurol. 38: 643; 1995) or of the soluble tau protein (Vadermeeren etal., Detection of tau proteins in normal and Alzheimer's diseasecerebrospinal fluid with a sensitive sandwich enzyme-linkedimmunosorbent assay, J. Neurochemistry 61: 1828-1834; 1993) in thecerebrospinal fluid. In the U.S. Pat. No. 5,593,846, a method fordetermining the concentration of soluble amyloid-β protein has beendescribed. However, the actual pathological component, the proteindepositions themselves, cannot be measured with this method.

[0004] After having concentrated cerebrospinal fluid, Townsend foundstructures therein which can be stained with the long known dyethioflavine S which is specific for protein aggregates (Townsend et al.,1987, Neurochemical Pathology, 6, 213-229).

[0005] In the corresponding U.S. Pat. No. 5,486,460, a method fordiagnosing Alzheimer's disease is described in which concentratedcerebrospinal fluid is plated on a glass surface and, after havingdried, stained with thioflavine S. However, this method is inconvenientin practice. In addition, the staining method with thioflavine S is notunambiguous for pathological protein depositions linked with Alzheimer'sdisease (see above). The method described did not meet with any furtherattention in the relevant art, and neither did the correspondingpublication.

[0006] In U.S. Pat. No. 5,434,050, Maggio describes a method fordiagnosing Alzheimer's disease by associating Aβ peptides to Aβaggregates, which are present as a solid bound structure, e.g., as abrain slice material. However, this diagnostic method can be practicedonly post mortem, as in the living patient, it would require a severesurgical intervention for obtaining brain biopsy material.

[0007] Therefore, it has long been desired to perform a measurement inbody fluids, such as cerebrospinal fluid.

[0008] Surprisingly, the above object is achieved by a diagnostic methodhaving the features of claim 1.

[0009]FIG. 1 shows a scheme of the method according to the invention fordiagnosing pathological protein depositions.

[0010]FIG. 2 shows the results of an experiment for the detection ofprion-protein aggregates by fluorescence correlation spectroscopy.

[0011]FIG. 3 shows the results of an experiment for the detection ofisolated prions from tissue samples by fluorescence-labeled solubilizedprion proteins (PrP-Cy2).

[0012]FIG. 4 shows the sensitivity of the homologous association offluorescence-labeled solubilized prion proteins (PrP-Cy2) toprion-protein aggregates from tissue samples and the influence of SDSconcentration.

[0013]FIG. 5 shows the homologous detection of peptide aggregates, whichrepresent the majority of amyloid depositions in Alzheimer's disease,with fluorescence-labeled soluble Aβ(1-42) peptide.

[0014]FIG. 6 shows the results of an experiment for the heterologousdetection of peptide aggregates, which represent the majority of amyloiddepositions in Alzheimer's disease, with fluorescence-labeledsolubilized prion protein (PrP-Cy2).

[0015]FIG. 7 shows the results of an experiment for the specificdetection of aggregates in the cerebrospinal fluid of patients for whomAlzheimer's disease has been diagnosed.

[0016]FIG. 8 shows a scheme of the screening for active substances.

[0017]FIG. 9 shows the cross-correlation function offluorescence-labeled prion protein (90-231).

[0018]FIG. 10 shows the association of a probe of recombinant prionprotein and a monoclonal antibody to a prion-protein aggregate.

[0019]FIG. 11 shows the frequency distribution of the fluorescencephotons for CJD-positive (left) and CJD-negative (right) samples ofcerebrospinal fluid.

[0020]FIG. 12 shows the frequency of fluorescence photons from channelswith more than 150 counts/channel in CJD-positive and CJD-negativesamples of cerebrospinal fluid.

[0021]FIG. 13 shows the influence of Congo red on the association of thespecific probe Aβ1-42CY2.

[0022] The method according to the invention for the diagnosticdetection of diseases comprises the measurement of the association ofsubstructures of the pathological protein depositions, structuresforming pathological protein depositions, structures corresponding topathological protein depositions and/or pathological protein depositionsas a probe to substructures of the pathological protein depositions,structures forming pathological protein depositions, structurescorresponding to pathological protein depositions and/or pathologicalprotein depositions as targets.

[0023] The method according to the invention is characterized in thatthe target is detected in liquid phase wherein, in the case of detectingAlzheimer's disease, the liquid phase is obtained from body fluids or isitself a body fluid. The association of the probe to the target ismeasured before self-aggregation of the probe predominates.

[0024] If the association of the probe to the target recedes into thebackground before self-aggregation of the probe, a reliable measurementcan no longer be ensured. Usually, measuring times in the range ofminutes and hours are possible, from which the method appears to besufficient for use in routine laboratories. The times in which themeasurements are performed naturally depend on the respective measuringconditions, but can be established relatively easily in preliminaryexperiments. As parameters which can influence the measuring time, theremay be mentioned, in particular, the concentrations of the targets/ofthe probe. For example, if the probe concentrations are rather high ascompared to the target concentration, self-aggregation of the probeswill start sooner than it would be the case if the probes were presentin a low concentration or if the probe concentration even was of asimilar order of magnitude as the target concentration or lower.

[0025] Measuring times of less than one hour, especially less than 30minutes, are preferred. Such measuring times are advantageous forperforming diagnoses in a routine manner.

[0026] According to the invention, the probe and/or the targetpreferably have a detectable property.

[0027] Preferably, the substructures of the pathological proteindepositions are monomeric or oligomeric units of pathological proteindepositions. The substructures may also be homologous, especiallystructurally homologous, with monomeric or oligomeric units ofpathological protein depositions.

[0028] The probe may be derived from the same (homologous detection) ora different type (heterologous detection) of pathological proteindepositions from that used as the target. Thus, for example, a structureforming pathological protein depositions, a structure corresponding topathological protein depositions and/or pathological protein depositionsthemselves derived from a prion disease, such as scrapie or BSE, may beemployed as the target while the probe may also be derived, for example,from other structures than those mentioned. Thus, it is possible, forexample, to measure the association of substructures (probe) derivedfrom amyloid depositions from BSE to protein depositions connected withAlzheimer's disease.

[0029] The substructures employed according to the invention maypreferably be obtained by treating pathological protein depositions withphysical means, such as ultrasonication, the action of temperaturechanges, chemical means, such as treatment with solutions of differentionic strengths, treatment with solutions of chaotropic ions, treatmentwith detergents and/or enzymes, especially proteases. Thus, it ispossible, for example, to disrupt amyloid plaques from scrapie-afflictedneuronal tissue into substructures by enzymatic degradation followed byultrasonication in the presence of detergents; subsequently, thesesubstructures may again form fibrillar structures. The substructures ofthe pathological protein depositions may also be recombinant proteins,protein fragments or peptides which may have sequence homologies withthe corresponding amyloid plaques of various origins and types.Structures corresponding to pathological protein depositions may also beused as probes. Such structures mimic regions of the actual pathologicalprotein depositions and associatively interact with the targets.

[0030] Structures forming pathological protein depositions may be usedas targets to which the association of probes is measured. This meansstructures which themselves are not the actual pathological proteindepositions, but monomeric or oligomeric units of the pathologicalprotein depositions, or larger aggregates of the substructures of thepathological protein depositions the association of which is to bemeasured. As an alternative, the pathological protein depositionsthemselves may also be used.

[0031] The probe and/or the target preferably have a detectableproperty. Said detectable property is either intrinsic to the abovementioned structures or protein depositions, especially to thesubstructure, or it can be introduced later. Said at least onedetectable property is established, in particular, by physical methods,preferably by spectroscopy. As a detectable property of the abovementioned structures or protein depositions, especially thesubstructure, their size or dimensions may be used, for example.Properties such as the structure, measurable by circular dichroism,optical properties such as luminescence, fluorescence or absorption mayalso be employed for measuring the association of the probes to thetargets. Thus, for example, both the intrinsic fluorescence ofstructures and the fluorescence of later labeled structures havingfluorescent properties can be used.

[0032] To increase the specificity or selectivity of the diagnosticdetection, other substances which interact with the targets may be used.For example, it is possible to allow substances having an affinity forthe targets, for example, antibodies or avidin/biotin, to interact withthe targets.

[0033] As the methods for measuring said at least one detectableproperty of the above mentioned structures or associations, there may bepreferably employed fluorimetric methods, such as confocal fluorescencespectroscopy, fluorescence correlation spectroscopy (FCS), FCS incombination with cross-correlation, with their respective appropriateevaluation methods. Included herein by reference are DE 44 38 341 andWO-A-96/13744, WO-A-94/16313, and EP-A-96 116 373 and EP-A-97 109 353.Especially the use of FCS cross correlation is of advantage because thespecificity of the method can be improved by this method. False positivedetections of aggregates in biological media can be suppressed, inparticular, by the use of different substructures as probes or thecombined use together with other probes for pathological proteindepositions, such as specific antibodies.

[0034] It may be preferred to recur to a method of “screening” themeasuring solution through a confocal volume element moved through themeasuring solution in order to increase the sensitivity of detection andto increase the rate of the analyses.

[0035] When cross correlation is used, two species having differentfluorescence are respectively observed.

[0036] The detectable properties are generated, for example, byfluorescence labels which may be low-molecular weight groups, but alsohigh-molecular weight groups. Thus, for example, labeled antibodieswhich are bound to the targets may label the latter. Then, the bindingof probes can be appropriately measured. Different probes may be labeledin different colors, for which cross correlation is then performed. Itis also possible to provide the probes with appropriate labels which areeither low-molecular weight fluorescent ligands and/or correspondinglabel conjugates.

[0037] According to the invention, the pathological protein depositionsare preferably derived from amyloid plaques which accompanyneurodegenerative diseases, such as Alzheimer's disease, bovinespongiform encephalopathy (BSE), Creutzfeldt-Jakob disease, scrapie,Huntington's chorea, Parkinson's disease, or from amyloid plaques fromorgans other than the neuronal system, such as protein depositionsassociated with diabetes. A suitable source for the pathological proteindepositions are organ extracts, preferably brain extracts of afflictedanimals. This may be, for example, prion-infected Syrian gold hamsters.The following survey lists diseases which are associated with amyloidphenomena: amyloid disease occurrence amyloid-forming proteinAlzheimer's disease neuronal amyloid-β protein transmissible spongiformneuronal prion protein encephalopathies Huntington's chorea* neuronalhuntingtin* Parkinson's disease neuronal synuclein* hereditary cerebralamyloid neuronal cystatin C angiopathy primary systemic amyloidosissystemic immunoglobulin (AL amyloidosis) reactive systemic amyloidosissystemic lipoproteins (AA amyloidosis) familial amyloid systemictransthyretin polyneuropathy type II diabetes pancreas islet amyloidpolypeptide injection-localized amyloidosis insulin medullary carcinomaof the thyroid gland calcitonin thyroid gland beta-2 microglobulinskeleton beta-2 microglobulin amyloidosis muscles hereditarynon-neuropathic systemic lysozyme amyloidosis Finnish hereditarysystemic systemic gelsolin amyloidosis

[0038] The substructures of the pathological protein depositions mayalso be recombinant proteins, protein fragments or peptides which havesequence homologies with amyloid plaques. Thus, for example,conservative amino acid substitutions in highly conserved regions can beas follows: any isoleucine, valine and leucine amino acid may beexchanged for any other of these amino acids, aspartate may be exchangedfor glutamate or vice versa, glutamine for asparagine or vice versa,serine for threonine or vice versa. Conservative amino acidsubstitutions in less highly conserved regions can be as follows: any ofthe amino acids isoleucine, valine and leucine may be exchanged for anyother of these amino acids, aspartate for glutamate or vice versa,glutamine for asparagine or vice versa, serine for threonine or viceversa, glycine for alanine or vice versa, alanine for valine or viceversa, methionine for any of the amino acids leucine, isoleucine orvaline, lysine for arginine or vice versa, any of the amino acidsaspartate or glutamate for any of the amino acids arginine or lysine,histidine for any of the amino acids arginine or lysine, glutamine forglutamate or vice versa, and asparagine for aspartate or vice versa.

[0039] As the source for material which is subjected to a diagnosticexamination by the method according to the invention, there may be used,in particular, body fluids, such as cerebrospinal fluid, blood, lymph,urine or secretions such as sputum. From the mentioned body fluids orsecretions, samples are taken and contacted with probes and incubatedfor measuring the association of structures forming pathological proteindepositions, structures corresponding to pathological proteindepositions and/or pathological protein depositions which could becontained in these samples. The presence of the pathological proteindepositions which are indicative of a related disease will then give apositive diagnostic signal, mediated by the association of the addedprobes. Body fluids are advantageous over tissues because they can besubjected to direct incubation whereas tissues must be first lysed as arule. This may be done, for example, by mechanical or chemical treatmentor combinations thereof.

[0040] The parameters established from the detectable properties of thementioned structures and depositions, especially substructures, may be,in particular, translational diffusion rates, rotational diffusionrates, lifetimes of excited states, polarization of radiation, energytransfer, quantum yield, number or concentration of particles, intensitydifferences. If the translational diffusion rate is established, it maybe desirable for slowly diffusing associates to detect them by multipledetection using a scanning process.

[0041] The load on the patient from the lumbal punction for obtainingcerebrospinal fluid is low. A significant increase in sensitivity can beachieved when a nucleated polymerization process is included astheoretically described by Jarret and Lansbury (Cell 73, 1055-1058,1993) for amyloid diseases. In this model, the aggregation of amyloiddepositions is classified into two kinetic reactions. In theself-aggregation kinetics of amyloid proteins into oligomeric subunits,the equilibrium is clearly on the side of the educts. Aggregates onlyform after an extended reaction time. A nucleation-dependentpolymerization ensues in which amyloid proteins associate to theaggregates already present. Here, the equilibrium is clearly on the sideof the products. Association occurs without a time delay. This timewindow between the two kinetic reactions should be useful for thedetection of natural protein depositions. After adding a probe which cansupport a polymerization process, in the case of the presence ofpathological protein depositions serving as nuclei, a clearlyaccelerated inclusion of the probe into such protein deposition shouldoccur as compared to the formation of the aggregates formed by theslower self-aggregation of the probe which, however, may not bedistinguishable from the former aggregates.

[0042] Especially the combination of nucleated association of amultitude of labeled probes to pathological protein depositions servingas the nuclei, with the extremely sensitive detection method by confocaloptics yields a correspondingly large amplification of the measuringsignal. Thus, in the cerebrospinal fluid obtained by lumbal punction(Neumeister et al., Klinikleit-faden Ladordiagnostik, Fischer-Verlag1998), individual molecules of the pathological protein depositionscould be unambiguously detected.

[0043] A screening method for detecting active substances for thetreatment of diseases associated with pathological protein depositions,which is analogous with the method according to the invention, is basedon the fact that the association of substructures of the pathologicalprotein depositions, structures forming pathological proteindepositions, structures corresponding to pathological proteindepositions and/or pathological protein depositions to substructures ofthe pathological protein depositions, structures forming pathologicalprotein depositions, structures corresponding to pathological proteindepositions as targets is measured in the presence of suspected activesubstances. As an alternative, association of substructures ofpathological protein depositions, structures forming pathologicalprotein depositions, structures corresponding to pathological proteindepositions and/or pathological protein depositions to substructures ofthe pathological protein depositions, structures forming pathologicalprotein depositions, structures corresponding to pathological proteindepositions as targets is first performed, and then the reversal of theassociation under the action of suspected active substances is observed.Then, those compounds are identified as active substances which arecapable of causing a reversal of the association in a significant way. Athird alternative is the measurement of the dissociation of proteindepositions themselves under the action of suspected active substances.

[0044] The method according to the invention is further illustrated bythe following Examples.

[0045] The scheme shown in FIG. 1 shows the experimental procedure forthe following Examples 1 to 6, 8 to 10.

[0046] Experiments 1 to 3 and 5 were performed with solubilized prionproteins while experiments 7 to 10 relate to recombinant prion protein(90-231).

[0047] The preparation of soluble (solubilized) prion proteins (PrP) waseffected by ultrasonication in 10 mM sodium phosphate, pH 7.2, 0.2% SDS,of prion aggregates isolated from infectious brain tissue of the Syriangold hamster (Riesner, D.; Kellings, K.; Post, K.; Wille, H.; Serban,H.; Groth, D.; Baldwin, M. A.; and Prusiner, S. B.; 1996, Journal ofVirology, Vol. 70, No. 3, pages 1714-1722). Depending on the energy ofultrasonication, a monomeric to oligomeric PrP fraction is formed whichwas fluorescence-labeled for the Examples described in the following (1to 3, 5) (Pitschke, M.; Post, K.; and Riesner, D.; 1997, Progress inColloid & Polymer Science, Vol. 107, pages 72-76). For fluorescencelabeling, the dye Cy2 (Amersham) was used.

[0048] Soluble Aβ(1-42) could be stabilized in 10 mM Na phosphatebuffer, pH 7.0, with 0.2% SDS (Examples 4, 6). The dye Cy2 (Amersham)was also used for the fluorescence labeling of Aβ.

[0049] For association measurements, the soluble, monomeric tooligomeric fluorescence-labeled proteins or peptides were incubated withamyloid aggregates (infectious prion particles: Examples 1 to 3; Aβ:Examples 4 to 5) with diluting the SDS to 0.2% SDS, and the fluorescencesignal was detected by fluorescence correlation spectroscopy.

EXAMPLE 1

[0050] Detection of Prion-protein Aggregates by Fluorescence CorrelationSpectroscopy (FCS)

[0051] Detection of infectious prion particles (PrP) by FCS through theassociation of fluorescence-labeled solubilized PrP.

[0052] Material

[0053] solubilized PrP-Cy2, about 25 ng/μl in 10 mM Na phosphate buffer,pH 7.0, 0.2% SDS; →(solution 1)

[0054] prion particles (about 200 ng/μl) in 10 mM Na phosphate buffer,pH 7.0; →(solution 2)

[0055] Performance

[0056] Two batches were perpared: A) 1 μl of solution 1 (1:10) B) 1 μlof solution 1 (1:10) 19 μl of Na phosphate buffer, 17 μl of Na phosphatebuffer, pH 7.0 pH 7.0 2 μl of solution 2 (1:100)

[0057] Each of the two charges was mixed, incubated for 1 minute,charged into a sample chamber and measured by FCS.

[0058]FIG. 2 shows the result of a measurement of fluorescence intensity(in relative units; RU) over a period of 60 seconds. In FIG. 2a, thefluctuation signal of the solubilized PrP-Cy2 is seen; it has a signalintensity between 110 and 130 RU. Evaluation of the signal usingautocorrelation yields a diffusion time of about 250 μs which istypically that of the monomeric protein. In FIG. 2b, a signal peakhaving a fluorescence intensity of about 950 RU is additionally seen; itis caused by the slow diffusion of a larger aggregate to which thepreviously solubilized fluorescence-labeled PrP-Cy2 is associated. Sincethis is a single event, determination of the diffusion time or molecularweight by autocorrelation is not possible. An unambiguous signaldetection and assignment to individual aggregates is successfullyachieved, however, through the occurrence of isolated peaks with adrastic increase in fluorescence intensity.

EXAMPLE 2

[0059] Homologous Detection of Isolated Prions from Tissue Samples UsingFluorescence-labeled Solubilized Prion Proteins (PrP-Cy2)

[0060] Detection of infectious aggregates of the prion proteins (PrProds) in brain extracts after labeling with fluorescence-labeledsolubilized prion proteins (PrP-Cy2) using FCS.

[0061] Material

[0062] solubilized PrP-Cy2, about 25 ng/μl in 10 mM Na phosphate buffer,pH 7.0, 0.2% SDS; →(solution 1)

[0063] brain extract from non-infected (solution 2) and prion-infectedSyrian gold hamsters (solution 3)

[0064] Performance

[0065] Two batches were prepared: A) 1 μl of solution 1 (1:10) B) 1 μlof solution 1 (1:10) 17 μl of Na phosphate buffer, 17 μl of Na phosphatebuffer, pH 7.0 pH 7.0 2 μl of solution 2 (1:10) 2 μl of solution 3(1:10)

[0066] Each of the two charges was mixed, incubated for 1 minute,charged into a sample chamber and measured by FCS.

[0067] As a control, solubilized PrP-Cy2 without addition of brainextract was measured in the same concentration.

[0068] In FIG. 3, the peak frequency for the individual charges isplotted as a graphical representation. The number of the peaks caused bythe diffusion of high-molecular weight aggregates through the confocalvolume element (see Example 1) was detected. It can be seen that theprion-infected tissue sample has a significantly higher peak frequencyas compared to the sample with non-infected brain extract.

[0069] Detection of infectious material through the association ofsolubilized PrP-Cy2 to existing prions is possible with thisexperimental design even if the peak frequency shows a background inexaminations of non-infected tissue samples.

EXAMPLE 3

[0070] Sensitivity of the Homologous Association of Fluorescence-labeledSolubilized Prion Proteins (PrP-Cy2) and of Prion-protein AggregatesIsolated from Tissue Samples

[0071] The sensitivity of the homologous association of PrP-Cy2 toprions isolated from infectious brain tissue is analyzed by FCS.

[0072] Material

[0073] solubilized PrP-Cy2, about 25 ng/μl in 10 mM Na phosphate buffer,pH 7.0, 0.2% SDS; →(solution 1)

[0074] PrP rods (about 200 ng/μl) in 10 mM Na phosphate buffer, pH 7.0;→(solution 2)

[0075] Performance

[0076] Two series of experiments were prepared: A) 1 μl of solution 1(1:10) B) 1 μl of solution 1 (1:10) 17 μl of Na phosphate buffer, 17 μlof Na phosphate buffer, pH 7.0 pH 7.0; 0.2% SDS 2 μl of solution 2 (1:x;PrP 2 μl of solution 2 (1:x; PrP quantity see FIG. 4) quantity see FIG.4)

[0077] The quantity of PrP rods in the experimental charge was reducedstepwise down to the detection limit. The measurement in 0.2% SDS servedas a control.

[0078] Every batch was mixed, incubated for 1 minute, charged into asample chamber and measured by FCS.

[0079] The peak frequency expressed in peaks per 10 min of measuringtime was plotted against the PrP quantity in the experimental charge inFIG. 4. Over the measured concentration range of 40 ng to 400 pg, adirect linear dependence of the peak frequency can be seen. Thus,evaluation of the number of peaks enables a direct quantification of theprion concentration in the sample to be measured. In 0.2% SDS,incorporation in existing PrP structures is not possible.

EXAMPLE 4

[0080] Homologous Detection of Peptide Aggregates, which Represent theMajority of Amyloid Depositions in Alzheimer's Disease, withFluorescence-labeled Soluble Aβ(1 -42) peptide

[0081] Detection of aggregates from Alzheimer's disease withfluorescence-labeled soluble Aβ(1-42) using FCS, to answer the questionof whether the detection method employed in Examples 1 to 3 can betransferred to other diseases involving pathological protein depositionsand is thus suitable for detection of any amyloid structure.

[0082] Material

[0083] soluble Aβ peptide 1-42, fluorescence-labeled with Cy2, 100 ng/μlin 10 mM Na phosphate buffer, pH 7.0, 0.2% SDS; →(solution 1)

[0084] Aβ amyloid fibrils (90 ng/μl) in 10 mM Na phosphate buffer, pH7.0; →(solution 2)

[0085] Performance

[0086] Two batches were prepared: A) 1 μl of solution 1 (1:10) B) 1 μlof solution 1 (1:10) 10 μl of solution 2 10 μl of solution 2 9 μl of Naphosphate buffer, 9 μl of Na phosphate buffer, pH 7.0 0.2% SDS, pH 7.0

[0087] Each of the two charges was mixed, incubated for 1 minute,charged into a sample chamber and measured by FCS.

[0088] As a control, soluble Aβ(1-42)-Cy2 was measured in the sameconcentration.

[0089] In FIG. 5, the results of the experiment are summarizedgraphically. The evaluation of peak frequency (cf. 1-3) shows that withincubation in 0.01% SDS, labeling of Aβ fibrils (SDS concentrationidentical with that used for PrP aggregates), which represent the majorcomponents of amyloid depositions in Alzheimer's disease, is possible bythis method. Incorporation of Aβ-Cy2(1-42) in the presence of anincreased SDS concentration (0.1%) is not possible. The labeling of Aβwhich is present in a soluble (rather than fibrillar) structure is notpossible either. The experiment shows that the detection of amyloiddepositions accompanying Alzheimer's disease is possible.

EXAMPLE 5

[0090] Heterologous Detection of Peptide Aggregates, which Represent theMajority of Amyloid depositions in Alzheimer's Disease, withFluorescence-labeled Solubilized Prion Proteins (PrP-Cy2)

[0091] Detection of Aggregates from Alzheimer's disease withfluorescence-labeled soluble PrP-Cy2 using FCS, to answer the questionof whether a heterologous labeling with a fluorescence-labeled solubleprobe not identical with the sample to which association is to takeplace is possible.

[0092] Material

[0093] solubilized PrP-Cy2, about 25 ng/μl in 10 mM Na phosphate buffer,pH 7.0, 0.2% SDS; →(solution 1)

[0094] Aβ(1-42) amyloid fibrils (90 ng/μl) in 10 mM Na phosphate buffer,pH 7.0; →(solution 2)

[0095] Aβ(1-42), non-fibrillar (90 ng/μl) in 10 mM Na phosphate buffer,pH 7.0, 0.2% SDS; →(solution 3)

[0096] Aβ(1-40) amyloid fibrils (90 ng/μl) in 10 mM Na phosphate buffer,pH 7.0; →(solution 4)

[0097] Aβ(1-40), non-fibrillar (90 ng/μl) in 10 mM Na phosphate buffer,pH 7.0, 0.2% SDS; →(solution 5)

[0098] Performance

[0099] The following batches were prepared: Aβ peptide (1-42): A) 1 μlof solution 1 (1:10) B) 1 μl of solution 1 (1:10) 2.5 μl of solution 22.5 μl of solution 3 16.5 μl of Na phosphate buffer, 16.5 μl of Naphosphate pH 7.0 buffer, pH 7.0 C) 1 μl of solution 1 (1:10) 2.5 μl ofsolution 2 16.5 μl of Na phosphate buffer, pH 7.0, 0.2% SDS Aβ peptide(1-40): A) 1 μl of solution 1 (1:10) B) 1 μl of solution 1 (1:10) 2.5 μlof solution 4 2.5 μl of solution 5 16.5 μl of Na phosphate buffer, 16.5μl of Na phosphate pH 7.0 buffer, pH 7.0 C) 1 μl of solution 1 (1:10)2.5 μl of solution 4 16.5 μl of Na phosphate buffer, pH 7.0, 0.2% SDS

[0100] Every batch was mixed, incubated for 1 minute, charged into asample chamber and measured by FCS.

[0101] As a control, soluble PrP-Cy2 was measured in the sameconcentration with 0.01% SDS.

[0102] The peak frequency (cf. Example 2) was measured for each of theabove mentioned batches (FIG. 6). It is found that both Aβ(1-42) andAβ(1-40) can be distinctly labeled with PrP-Cy2 if they are present in afibrillar structure and if the labeling reaction takes place at 0.01%SDS. When association is effected to non-fibrillar structures or the SDSconcentration is increased, only a low background signal can be seen.

[0103] This result shows that the labeling of amyloid protein aggregatesdoes not forcibly require that the soluble fluorescence-labeled proteinemployed for labeling be identical with the protein forming theaggregate.

EXAMPLE 6

[0104] Detection of Aggregates in the Cerebrospinal Fluid of PatientsSuffering from Alzheimer's Disease

[0105] Specific detection of aggregates associated with Alzheimer'sdisease. Thus, cerebrospinal fluid (spinal fluid) is used as a sample inwhich a specific distinction can be made between patients suffering fromAlzheimer's disease and a healthy control group.

[0106] Material

[0107] soluble Aβ peptide 1-42, fluorescence-labeled with Cy2, 100 ng/μlin 10 mM Na phosphate buffer, pH 7.0, 0.2% SDS; →(solution 1)

[0108] cerebrospinal fluid from afflicted patients and control fromnon-afflicted subjects

[0109] Performance

[0110] The following batches were prepared:

[0111] 2 μl of solution 1 (1:10)

[0112] 18 μl of cerebrospinal fluid from patients

[0113] Without further treatment or concentration, the cerebrospinalfluid samples were directly incubated with fluorescence-labeledAβ(1-42)-Cy2 (0.1 ng/μl) at 0.02% SDS and immediately measured by FCSfor 20 min (FIG. 7). The detected aggregates in cerebrospinal fluidsamples from patients afflicted with Alzheimer's dementia (AD) as judgedby psychiatric diagnostic criteria and those from non-afflicted controlsubjects were compared. It is found that all patients afflicted withAlzheimer's dementia have a significantly higher signal intensity in thecerebrospinal fluid as compared to the corresponding control group whichhad a similar age structure. The patient designated as CAA has thehighest signal intensity of all patients subjected to diagnosis. Thispatient is afflicted with congophile angiopathy, a special form ofAlzheimer's disease in which deposition of amyloid-β peptides occurs inthe capillaries of the cerebral vessels, the migration of which isthereby gradually destroyed. In this disease, the most massivetransgression of Aβ aggregates into the cerebrospinal fluid seems totake place, which results in the large increase of signal intensity. Themeasuring results of the control group show that aggregates are detectedhere as well in isolated cases. This result is also shown by the probealone (Aβ(1-42)-Cy2); it is due to self-aggregation effects occurringduring the 20 minutes' measuring time or due to existing Aβ aggregates.The measuring results of the probe were achieved in 0.01% or 0.2% SDS.

EXAMPLE 7

[0114] Coaggregation of Two Fluorescence-labeled rPrP Probes to rPrP(90-231)

[0115] Recombinant prion protein (90-231) (referred to as rPrPhereinafter) which is homologous with the protease-resistant portion ofhamster PrP was labeled with amino-reactive fluorescence dyes of theexcitation wavelengths 488 nm (Oregon Green, Molecular Probes) and 633nm (Cy5, Amersham). The protein concentrations and labeling ratio weredetermined by absorption measurements at 280 nm, 496 nm and 650 nm. Thelabeling efficiency was 10% (rPrP-Cy5) and 4% (rPrP-OrG) for equimolaraddition of the amino-reactive fluorescence dye.

[0116] Aggregation experiments: Equimolar amounts of the two labeledrPrP probes were mixed with a 25fold excess of unlabeled rPrP, thebuffer containing 0.2% SDS. The aggregation process was started bydiluting to an SDS concentration of 0.01% and an rPrP totalconcentration of 50 nM.

[0117] The aggregation process was followed by repeatedly recordingautocorrelation and cross-correlation curves in two-color crosscorrelation FCS equipment (ConfoCor prototype, Zeiss). The correlationcurves were shaped by a three-dimensional diffusion model wherein adiffusion parameter τrg was used for the cross-correlation curves.Fitting was performed using a model for spherical molecules.

[0118] Reduction of the SDS concentration causes a rapid increase of thecross-correlation amplitude which is proportional to the concentrationof the particles into which both red and green labeled PrP wereincorporated. In the presence of 0.2% SDS, a cross-correlation productcould not be detected. In a few minutes, the oligomer concentrationrises up to a plateau and then slowly decreases through the formation ofhigher aggregates.

[0119]FIG. 9 (left) shows the cross-correlation function offluorescence-labeled rPrP(90-231), c(rPrP)=50 nM, c(rPrP-Cy5),c(rPrP-OrG)=2 nM in PBS, 25° C.+0.01% SDS. The curves have the followingmeanings: a) smaller dots: 1 min incubation time; b) short dashes: 6min; c) larger dots: 20 min; d) dashed line: 30 min; e) solid line: 130min; f) small dots: reference sample, 0.2% SDS.

[0120]FIG. 9 (right) shows the plot of the cross-correlation amplitudein the course of aggregation (squares). The formation of particles whichhave incorporated both red and green fluorophores can be described by asecond order reaction: A_(i+)B_(j→)AB_(ij), k=5·10⁶ M⁻¹.

EXAMPLE 8

[0121] Association of rPrP Probes to Existing rPrP Aggregates

[0122] Preaggregated rPrP in concentrations of from 0.2 to 20 ng/μl wasadded to the aggregation batch of Example 7. The rPrP aggregates wereproduced by incubation of rPrP at an SDS concentration of 0.01% for 1 hat 25° C. (c(rPrP)=100 ng/ml). The association process was followed bythe consecutive recording of autocorrelation and cross-correlationcurves

[0123] The association of labeled rPrP to the aggregates could beobserved within a time in the range of a few minutes. This process wascharacterized by the occurrence of large fluorescence peaks in the rawsignal. The cross-correlation curves were dominated by a componenthaving a high diffusion time (>5 ms).

EXAMPLE 9 Recognition of rPrP Aggregates by the Cross-correlation of TwoProbes: Monoclonal Antibody (mAB)+rPrP

[0124] For the specific recognition of prion protein, various monoclonalantibodies are available which are distinguished by their specificityand binding strength. Particularly suitable for the recognition ofpathogenic PrP in samples of CJD patients is an antibody developed by B.Oesch (Korth et al., Nature, Nov. 6, 1997, 390 (6655): 74-7) whichspecifically binds the pathogenic form of the prion protein. Thecombination of specific antibody binding with the coaggregation of PrPprobes offers an increased specificity as compared to the singlelabeling of the pathogenic aggregates.

[0125] The monoclonal antibody IgM 15b3 was labeled with a fluorescencedye at its amino functions by analogy with the PrP probes. The labeledantibody was employed in a final concentration of 20 nM. For the mixeduse of rPrP and antibody probes, it is desirable that the bindingconditions be suitable for both probes. At an SDS concentration of0.01%, both coaggregation of the rPrP probe and antibody binding can beobserved. Entry of a labeled aggregate into the focus produces afluorescence peak. At an appropriate size, the peak signal dominates thecross-correlation curve.

[0126]FIG. 10 shows the association of the rPrP probe and mAB to an rPrPaggregate. Top: sweep of the cross-correlation fluorescence signal.Bottom: two-color cross-correlation curve. The course of the curve isdominated by the large aggregate at the beginning of the sweep. Theconcentrations are c(rPrP)=50 nM, c(rPrP-Cy2)=2 nM, c(mAB-Cy5)=20 nM.

EXAMPLE 10

[0127] Peak Analysis of the Fluorescence Raw Data

[0128] The association of many probe molecules to a protein targetgenerates highly labeled aggregates (>10 incorporated probes) whichprotrude before the background signal of the free probe by at least afactor of two. The amount of aggregates present in the sample can bedetermined by the number of peaks. Thus, with measuring times of 15 min,aggregates in concentrations of down to femtomolar can be detected.

[0129] One application is the detection of PrP aggregates in thecerebrospinal fluid of CJD patients. Three CJD-negative and threeCJD-positive cerebrospinal fluid samples were examined. Adifferentiation between CJD-positive and CJD-negative samples could bemade by the number of events with high fluorescence bursts.

[0130] Experimental conditions: Cy2-labeled rPrP probe was added to acerebrospinal fluid sample in the presence of 0.02% SDS so that probeconcentration was 10 nM. The measurement was effected at 22° C. for 15min.

[0131] Data acquisition: The samples were measured in a ConfoCor FCSdesign. Excitation was effected at a wavelength of 488 nm (Ar⁺ laser)with a power density of 5·10⁴ W/cm². The emitted fluorescence light wasfocused by a microscope objective (40×/1.2 NA, Zeiss) and confocallyimaged onto an avalanche photodiode. The latter acts as a photon counterwhich yields one signal pulse for each photon. The signal pulses weretransmitted through a signal divider and evaluated in parallel on ahardware correlator card and recorded on a scaler card with a channelwidth of 250 μs. The total measuring time was 15 min which correspondsto a total of 3.6 million channels.

[0132] Data evaluation: The evaluation was performed according to thenumber and height of the fluorescence peaks. A distribution of thenumber of photons per channel is shown in a histogram.

[0133]FIG. 11 shows the frequency distribution of the fluorescencephotons per measuring channel for a channel width of 250 μs andconcentration of the probe rPrP−Cy2=20 nM; left: CJD-positivecerebrospinal fluid sample, right: CJD-negative cerebrospinal fluidsample.

[0134] The height of the peaks represents the number of photons whilethe number of channels is a measure of the integral over the area of allpeaks. In the CJD-positive samples, a higher proportion of largefluorescence peaks is observed. By setting a threshold value, a cleardistinction can be made between CJD-positive and CJD-negative samples.

[0135]FIG. 12 shows the frequency of fluorescence photons from channelswith more than 150 counts/channel in CJD-positive (CJD+)/CJD-negative(CJD−) cerebrospinal fluid samples at a channel width of 250 μs and aconcentration of the probe rPrP-Cy2 of 20 nM. p315 et. are internalidentification numbers of the patients.

[0136] In the two-color design, a coincidence analysis can be made bymultiplying the red by the green signal in order to recognize peaks fromaggregates labeled with probes of both colors. By a refined peak-burstanalysis, the separation of large peaks from the signal of the probecould be further improved.

EXAMPLE 11

[0137] Congo Red as a Model for the Screening Method

[0138] Congo red is known for its inhibiting effect on fibrillogenesis.

[0139] The original measurement (20 measurements each with one minute ofmeasuring time) in the cerebrospinal fluid of a patient afflicted withAlzheimer's dementia is represented in FIG. 13.

[0140]FIG. 13 shows the influence of Congo red on the association of thespecific probe Aβ-42-Cy2 to synthetic Aβ1-42 aggregates. In the FCS, adecrease of the signal intensity of the homologous association to 20%can be observed upon the addition of 1 mM Congo red.

[0141] Upon the addition of 1 mM Congo red (bottom), a significantdecrease of the detectable peaks as compared to the control batch (top)can be observed. Because of the fact that Congo red absorbs in the rangeof the excitation wavelength, a different scaling of the measurement hasbeen done for samples with and without Congo red.

[0142] This shows that substances which inhibit fibrillogenesis can beidentified in FCS.

1. A method for the diagnostic detection of diseases associated withprotein depositions (pathological protein depositions) by measuring anassociation of substructures of the pathological protein depositions,structures forming pathological protein depositions, structurescorresponding to pathological protein depositions and/or pathologicalprotein depositions as a probe; to substructures of the pathologicalprotein depositions, structures forming pathological proteindepositions, structures corresponding to pathological proteindepositions and/or pathological protein depositions as targets;characterized in that the target is detected in liquid phase wherein, inthe case of detecting Alzheimer's disease, the liquid phase is obtainedfrom body fluids or is itself a body fluid; with the proviso that theassociation of the probe to the target is measured beforeself-aggregation of the probe predominates.
 2. The method according toclaim 1, wherein said substructures of the pathological proteindepositions, structures forming pathological protein depositions,structures corresponding to pathological protein depositions and/orpathological protein depositions have at least one detectable property.3. The method according to claim 1 and/or 2, wherein said substructuresof the pathological protein depositions having at least one detectableproperty are monomeric or oligomeric units of pathological proteindepositions, or the substructures are homologous with monomeric oroligomeric units of pathological protein depositions.
 4. The methodaccording to any of claims 1 to 3, wherein said probe and said targetare derived from the same or from different types of pathologicalprotein depositions.
 5. The method according to any of claims 1 to 4,wherein said substructures can be obtained by treating pathologicalprotein depositions with physical means, such as ultrasonication, theaction of temperature changes, or with chemical means, such as treatmentwith solutions of different ionic strengths, treatment with solutions ofchaotropic ions, treatment with detergents and/or enzymes, especiallyproteases.
 6. The method according to any of claims 1 to 5, wherein saidat least one detectable property is either intrinsic to thesubstructure, or it is introduced later.
 7. The method according to anyof claims 1 to 6, wherein other substances which will interact with thetargets are used to increase the specificity or selectivity of thediagnostic detection.
 8. The method according to any of claims 2 to 7,wherein said at least one detectable property is size or dimension,molecular weight, structure, circular dichroism, optical properties suchas luminescence, especially fluorescence, absorption, radioactivity. 9.The method according to any of claims 2 to 8, wherein the measurement ofsaid at least one detectable property is performed by physical methods,especially spectroscopic methods.
 10. The method according to any ofclaims 2 to 9, wherein the measurement of said at least one detectableproperty is performed by fluorimetric methods, such as confocalfluorescence spectroscopy, fluorescence correlation spectroscopy (FCS),FCS in combination with cross-correlation, with their respectiveappropriate evaluation methods.
 11. The method according to any ofclaims 1 to 10, wherein said pathological protein depositions areamyloid plaques and/or neurofibrillar filaments (NFT) which are derivedfrom the neuronal system and are associated with neurodegenerativediseases, such as Alzheimer's disease, transmissible spongiformencephalopathy, Huntington's chorea, Parkinson's disease, hereditarycerebral amyloid angiopathy.
 12. The method according to any of claims 1to 10, wherein said pathological protein depositions are, in particular,amyloid plaques derived from organs other than the neuronal system andare associated with diseases such as, in particular, primary systemicamyloidosis (AL amyloidosis), reactive systemic amyloidosis(AAamyloidosis), familial amyloid polyneuropathy, type II diabetes,injection-localized amyloidosis, medullary carcinoma of the thyroidgland, beta-2 microglobulin amyloidosis, hereditary non-neuropathicamyloidosis, Finnish hereditary systemic amyloidosis.
 13. The methodaccording to any of claims 1 to 12, wherein said probes are recombinantproteins, protein fragments or peptides having sequence homologies withamyloid plaques and/or neurofibrillar filaments.
 14. The methodaccording to any of claims 1 to 13, wherein samples are taken from bodyfluids, such as cerebrospinal fluid, blood, lymph, urine, or secretionssuch as sputum, or tissue and contacted with probes for the diagnosticdetection of any diseases, incubated, and the association of the probesto targets is measured.
 15. The method according to any of claims 2 to14, wherein parameters such as translational diffusion rates, rotationaldiffusion rates, lifetimes of excited states, polarization of radiation,energy transfer, quantum yield, number or concentration of particles,intensity differences are established from said detectable properties.